This invention relates generally to the field of molecular biology of telomere and telomere associated proteins, and the maintenance of telomere structure. More specifically, this invention relates to a novel protein that shares three domains of homology with the telomerase associated protein Tankyrase I.
Recent research has described what may be a key switch in the control of cellular aging. The telomeres at chromosome ends are made up of multiple repeats of the DNA sequence TTAGGG, which are thought to stabilize the chromosome during replication. Telomeres shorten each time the cell divides, and cells become senescent when the telomeres are too short to protect the chromosome. But in some cells, including embryonic cells, an enzyme called telomerase rebuilds the telomeres after each division, extending the replicative capacity of the cell. (Bodnar et al., Science 279:349, 1998; Harley et al., Curr. Opin. Genet. Dev. 5:249,1995).
Regulation of telomerase activity is a complex process involving several protein components. Two such proteins have DNA binding activity, and are named telomeric repeat binding factors (TRF1 and TRF2). It is thought that TRF1 is involved in regulating telomere length, because overexpression of wild-type TRF1 makes telomeres shorter, while overexpression of a dominant-negative form of TRF1 makes telomeres longerxe2x80x94perhaps by affecting the access of telomerase to the chromosome terminus (van Steensel et al., Nature, 385:740, 1997). TRF1 promotes parallel pairing of telomeric tracts, apparently pairing in parallel homodimers that form filamentous structures on longer telomeric repeat arrays (Griffith et al., J. Mol. Biol. 278:79-88, 1998).
The role of TRF2 appears to be protection of the chromosome terminus, since expression of a dominant-negative form of TRF2 leads to chromosomexe2x80x94chromosome fusions. (Griffith et al., J Mol Biol. 278:79, 1998; Broccoli et al., Nature Genetics 17:231, 1997; van Steensel et al., Cell 92:401, 1998). This in turn leads to p53xe2x80x94and ATM-dependent apoptosis of the cell (Karlseder et al., Science 283:1321, 1999). TRF1 and TRF2 have been implicated in large duplex loops at the end of telomeres that may provide a general mechanism for telomere protection and replication (Griffith et al., Cell 97:503, 1999).
Smith et al. (Science 282:1484, 1998; Genomics 57:320, 1999; J. Cell Sci. 112:3649, 1999) have reported a novel protein that associates with TRF1, which they named xe2x80x9cTankyrasexe2x80x9d. A yeast two-hybrid screen was used with human TRF1 as bait, and yielded two overlapping cDNAs which provided the full-length sequence. Northern blot analysis revealed that multiple mRNAs were ubiquitously expressed in human tissues, with the highest amounts detectable in testes. It has been proposed that tankyrase interferes with the binding of TRF1 to telomeres, which in turn has an effect on telomere length. Tankyrase co-localizes with TRF1 at the ends of human chromosomes in metaphase and interphase, and also resides at nuclear pore complexes and centrosomes. Smith et al. reported that the gene for tankyrase is positioned at 17.6 cR10000 on human chromosome 8 with a LOD of 8.2 on the G3 map.
The molecular events involved in managing chromosome structure and regulating cell senescence are extremely complex. Each new protein found to participate in this process provides new opportunities for monitoring and intervening in some of the fundamental events of cell biology.
This invention provides a new human protein which is hereby designated Tankyrase II. This new protein shares three domains with the Tankyrase protein of Smith et al.: the ANK domain comprising 24 repeats of the ankyrin motif, the SAM domain thought to be involved in proteinxe2x80x94protein interaction, and the PARP domain that is responsible for the poly(ADP-ribose) polymerase activity. Tankyrase II further comprises has a new domain at the N-terminal, designated the GC domain, which has no known homologs.
One of the embodiments of this invention is an isolated polynucleotide having at least about 30 consecutive nucleotides contained in a human Tankyrase II encoding sequence, or that is contained in plasmids deposited under Accession No. 203919, or that hybridizes under stringent conditions to a Tankyrase II encoding sequence, but does not consist of the encoding sequence for human Tankyrase I or other previously known structurally related proteins, such as those having PARP activity. Another embodiment of this invention is an isolated polynucleotide having at least 100 consecutive nucleotides that is at least 90% identical to a Tankyrase II sequence, or contained in the deposited plasmids, but not in xcex-phage, Tankyrase I, or other previously known sequences. Certain polynucleotides of this invention encode a protein comprising a GC domain, a PARP domain, a SAM domain, or an ANK domain, or a protein that binds other telomere-associated proteins like TRF1, TRF2, TIN2, and Tankyrase I, or that ADP-ribosylates a target protein in the presence of NAD+. Polynucleotides of this invention can be used to obtain the encoded polypeptide, or to determine other polynucleotides that encode Tankyrase II-like protein.
Another embodiment of this invention is an isolated polypeptide comprising a sequence of at least 10 consecutive amino acids that is contained in Tankyrase II, or is contained in the deposited plasmids, but is not contained in any previously known peptide sequence. Another embodiment of this invention is an isolated polypeptide comprising a sequence of at least 25 consecutive amino acids that is at least 90% identical to a Tankyrase II protein sequence, or a protein sequence encoded in the deposited plasmids. Certain polypeptides of this invention comprise a GC domain, a PARP domain, a SAM domain, or an ANK domain, or have activity for binding other telomere-associated proteins like TRF1, TRF2, TIN2, and Tankyrase I, or ADP-ribosylate a target protein in the presence of NAD+.
A further embodiment of this invention is an isolated human Tankyrase II protein or fragment thereof, at least 10-fold higher in purity (or more) on a weight per weight basis than what occurs in natural sources.
Also embodied in this invention are polynucleotides encoding the polypeptides of this invention, and antibodies of any sort that bind specifically to the polypeptides of this invention. Some of the antibodies inhibit the catalytic activity of Tankyrase II; inhibit the binding of Tankyrase II to other telomere associated protein; or inhibit protein ribosylation mediated by Tankyrase II. Peptides can be obtained by expressing a polynucleotide of the invention in a suitable host cell. Also provided are means for obtaining any antibody of this invention, comprising immunizing an animal or contacting an immunocompetent particle with a polypeptide of this invention. Peptides of this invention can be isolated from a mixture by using an antibody as a specific adsorbant; conversely, antibodies of this invention can be isolated using a peptide epitope as a specific adsorbant.
A further embodiment of this invention is a method for ribosylating a target protein, comprising incubating the target protein with a peptide of this invention in the presence of NAD+.
Assay methods of this invention include determining Tankyrase II binding activity by incubating with a peptide of this invention under conditions where the protein can bind the peptide specifically to form a complex, and then correlating any complex formed with the presence or amount of the protein in the sample. The protein that has Tankyrase II binding activity can optionally be TRF1, TRF2, TIN2, or Tankyrase I.
Another assay method of this invention is for screening a test compound to determine an ability to affect Tankyrase II activity, comprising incubating the compound with containing a peptide of this invention and a conjugate binding ligand, and determining any effect of the test compound on complex formation. Another such method comprises incubating a test compound with a peptide of this invention, a potential target protein, and NAD+; then determining any effect of the test compound on the amount or rate of ribosylation of the target.
This invention also includes a method for modulating Tankyrase II expression in a cell, comprising contacting the cell with the polynucleotide of this invention such as an antisense polynucleotide, a ribozyme, or an inhibitory RNA under conditions where the polynucleotide can interfere with mRNA translation. Modulating Tankyrase II expression in turn is believed to modulate telomere length in the cell.
These and other embodiments of the invention will be apparent from the description that follows.